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Biomedical subjects

R Julien

Publications and source records attributed to R Julien.

At least 19 recordsLinked to original sources
PubMed1997

Asymmetrical distribution of cardiolipin in yeast inner mitochondrial membrane triggered by carbon catabolite repression.

Transmembrane asymmetry of cardiolipin in yeast was monitored during the switch from fermentative to gluconeogenic growth and the reverse. As soon as cells used ethanol as an electron donor to produce ATP by oxidative phosphorylation, rapid and abundant cardiolipin synthesis was observed on the matrix side of the inner mitochondrial membrane followed by a transverse rearrangement between the two leaflets. The cardiolipin distribution changed from about 20:80 (in/out) to 70:30 (in/out), and after translocation towards the outer leaflet it finally became 37:63 (in/out). At the same time, cytochrome c oxidase activity remained stable, then increased as a possible result of the topographical rearrangement. During the reverse process from gluconeogenic to fermentative growth, the amount of cardiolipin rapidly decreased by half, its bilayer distribution apparently changing to a monolayer organization before the 20:80 (in/out) asymmetry of repressed cells was re-established. Experimental impairment of cardiolipin topography by antibiotic inhibition of gene expression or in situ dissipation of mitochondrial membrane potential produced data that prove that the amount and transmembrane distribution of the phospholipid are two specific parameters of the mitochondrial inner membrane organization in both fermentative (2.2 fmol/cell and 20:80, in/out) and gluconeogenic (4.2 fmol/cell and 37:63, in/out) growing yeast cells. Finally, the inner mitochondrial membrane topography of cardiolipin appeared to be closely associated with the transmembrane redox potential.

Biological Transport↗
PubMed1997

Molecular cloning and expression of a bovine alpha(1,3)-fucosyltransferase gene homologous to a putative ancestor gene of the human FUT3-FUT5-FUT6 cluster.

Only one bovine gene, corresponding to the human cluster of genes FUT3-FUT5-FUT6, was found by Southern blot analysis. The cognate bovine alpha(1,3)-fucosyltransferase shares 67.3, 69.0, and 69.3% amino acid sequence identities with human FUC-T3, FUC-T5, and FUC-T6 enzymes, respectively. As revealed by protein sequence alignment, potential sites for asparagine-linked glycosylation and conserved cysteines, the bovine enzyme is an intermediate between FUC-T3, FUC-T5, and FUC-T6 human enzymes. Transfected into COS-7 cells, the bovine gene induced the synthesis of an alpha(1, 3)-fucosyltransferase enzyme with type 2 substrate acceptor pattern specificity and induced expression of fucosylated type 2 epitopes (Lex and sialyl-Lex), but not of type 1 structures (Lea or sialyl-Lea), suggesting that it has an acceptor specificity similar to the human plasma FUC-T6. However, no enzyme activity was detected in bovine plasma. Gene transcripts are detected on tissues such as bovine liver, kidney, lung, and brain. The type 2 sialyl-Lex epitope was found in renal macula densa and biliary ducts, and Lex and Ley epitopes were detected on the brush border of epithelial cells of small and large intestine, suggesting a tissue distribution closer to human FUC-T3, but fucosylated type 1 structures (Lea, Leb, or sialyl-Lea) were not detected at all in any bovine tissue. Analysis of genetic distances on a combined phylogenetic tree of fucosyltransferase genes suggests that the bovine gene is the orthologous homologue of the ancestor of human genes constituting the present FUT3-FUT5-FUT6 cluster.

Amino Acid Sequence↗
PubMed1997

Associated effects of divergent selection for residual feed consumption on reproduction, sperm characteristics, and mitochondria of spermatozoa.

Eighteen generations of divergent selection for residual feed intake have been completed in two Rhode Island Red lines of domestic fowl. The high intake R+ line and the low intake R- line cocks used to sire Generation 19 of the selection experiment have been compared for associated responses on fertility, hatching, and sperm quality. Evaluations of sperm samples were based on volume, cell concentration, biochemical parameters (pH, uric acid and protein concentrations), and motility and morphology of spermatozoa. Finally, individual spermatozoa were analyzed by flow-cytometry (FCM) using Rhodamine 123 (Rh123) and nonyl-acrydine-orange (NAO) specific fluorochromes to assess, respectively, overall mitochondrial activity and overall mitochondrial content. Hatchability of incubated eggs was 20 points higher for the R- line, mainly because unfertilized eggs were only 6 vs 30% in the R+ line. Early embryo mortality was also twice as high in the R+ line (21%). The ratio of Rh123 to NAO fluorescence was identical for both lines. This result suggests that there was no difference in the energy producing potential of the individual mitochondria. Therefore, the difference seen for both dyes between the two lines might be attributed to a difference in the quantity of mitochondrial inner membranes present in the cell (with 17% less for the R+ line). In the R+ line, the poor performance at fertilization and during early embryonic development was associated with lower production of motile spermatozoa, possibly in relation to a lower quantity of mitochondria in spermatozoa from R+ cocks. Although the female contribution to the differences between lines was not explored separately, results suggest that selection for residual feed intake may have altered some cellular function related to the production of energy in the R+ line.

Analysis of Variance↗
PubMed1997

Convenient chromatographic prepurification step before measurement of urinary cortisol by radioimmunoassay.

We applied various prepurification protocols (extraction with different solvents, liquid/solid separation on bonded silica media, Celite, and Sephadex LH20 chromatography) with a range of commercially available RIA kits to measure cortisol in urine samples. We then compared the results with the concentrations measured by a HPLC method validated with reference to isotope dilution gas chromatography-mass spectrometry. We conclude that chromatography on a commercial, prepacked diol minicolumn (Waters Sep-Pak Vac RC) in combination with dichloromethane extraction is a convenient and very effective purification step before RIA of urinary cortisol in patients not receiving corticoid medication. We tested numerous steroids for interference and found that free polar cortisol derivatives (hydroxylated or hydrogenated) could only partially account for the overestimations routinely encountered when free urinary cortisol concentrations are measured by direct RIA.

Adult↗
PubMed1996

Pre-Main-Sequence Star Candidates in the Bar of the Large Magellanic Cloud

Candidate pre-main-sequence stars were observed in the bar of the Large Magellanic Cloud during the search for dark matter in the galactic halo. Seven blue stars of apparent visual magnitude 15 to 17 had irregular photometric variations and hydrogen emission lines in their optical spectra, which suggested that these stars are pre-main-sequence stars of about 10 solar masses. These stars are slightly more massive and definitely more luminous than are Herbig AeBe pre-main-sequence stars in our own galaxy. Continued observations of these very young stars from another galaxy, which are probably at the pre-hydrogen-burning stage, should provide important clues about early stages of star formation.

Journal Article↗
PubMed1996

Production, analysis and bioactivity of recombinant vasoactive intestinal peptide analogs.

Recombinant vasoactive intestinal polypeptide (VIP) analogs were expressed in Escherichia coli as a fusion protein containing tandemly repeated multiple copies of a synthetic VIP gene joined to glutathione S-transferase. The encoded protein contains VIP units separated by a linker peptide, potentially excisable by a double cleavage with endoprotease factor Xa and hydroxylamine. Expression of different polyVIP genes, from 1 to 32 units, was detected and the production of a 16 VIP polymer was performed. MonoVIP analogs appended by 5 or 10 amino acids at their C terminus were released by factor Xa from this polymerized product. They were then submitted to hydroxylamine cleavage to remove the linker sequence to finally obtain a recombinant VIP analog devoid of any amino acid extension. The biological activity of the recombinant polyVIP and VIP analogs was tested. Although less efficient than the natural neuropeptide, some of these components bound to VIP receptor, activated adenylate cyclase in human colonic adenocarcinoma cells and displayed a relaxation activity on guinea pig tracheal rings.

Amino Acid Sequence↗
PubMed1996

Secretion kinetics of endo-N-acetyl-beta-D-glucosaminidase during vegetative growth of Myxococcus xanthus.

It was recently demonstrated that endo-N-acetyl-beta-D-glucosaminidases (ENGase) acting on N-glycosylproteins are produced by myxobacteria. In this study, it was shown that the secretion of ENGase during vegetative growth of Myxococcus xanthus was cell-density-dependent. The activity produced per cell increased up to 6 x 10(8) cells/ml and stabilized thereafter (maximum level). Two of the developmental mutants used in this study (bsgA and csgA) were locked for ENGase secretion into the maximum level regardless of cell density. To explain the pattern of ENGase secretion, we postulated the presence of a molecule that induces the enzyme until it reaches a proper concentration threshold. Although the chemical structure of this cell density signal was not determined during this study, its occurrence during vegetative growth of M. xanthus was strongly suggested by the results.

In Vitro Techniques↗
PubMed1996

Linear correlation between bacterial overexpression of recombinant peptides and cell light scatter.

Fusion of multiple copies of a test peptide leads to insoluble inclusion bodies. Their presence within bacteria increases either forward-angle light scattering or, to a lesser extent, right-angle light scattering. A linear correlation has been established between cell forward-angle scattering and the level of overexpression of atrial natriuretic peptide. The correlation is valid only for unlysed cells and is protein product specific.

Atrial Natriuretic Factor↗
PubMed1996

11 beta-Hydroxysteroid dehydrogenase deficit: a rare cause of arterial Hypertension. Diagnosis and therapeutic approach in two young brothers.

We report the clinical history and results of endocrine investigations in two brothers born to consanguineous parents, who presented with hypokalemia and arterial hypertension when they were aged 2 and 6 years. The hormonal serum assay results, including extremely low values for aldosterone and plasma renin activity, favored the existence of apparent mineralocorticoid excess. A diagnosis of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) deficiency was made, based on assays of the hydrogenated urinary metabolites of cortisol and cortisone, as well as of corticosterone and dehydrocorticosterone. Indeed we found a very low rate of urinary elimination of cortisone metabolites: tetrahydrogenated cortisone was reduced to between 0.10 and 30 mumol/24 h, which is 15-100 times lower than the normal rate; hexahydrogenated cortolones alpha and beta were found to be 7- to 20-fold lower than normal levels; and the 11-keto-17-ketosteroid derivatives of cortisone were also reduced. Urinary elimination of the cortisol-reduced metabolites 5 beta- and 5 alpha-tetrahydrogenated cortisol were slightly reduced or normal. These results argue in favor of a deficit in the enzyme 11 beta-HSD, which oxidizes cortisol into cortisone. A moderate defect in the conversion of cortisol into 5 beta-THF compared to normal conversion into 5 alpha-THF was also found. With respect to corticosterone metabolism, we demonstrated the presence of a defect in the oxidation of that steroid into dehydrocorticosterone, also due to the deficit in 11 beta-HSD. Arterial hypertension and hypokalemia were corrected by treatment with dexamethasone, concomitantly with correction of the low aldosterone and plasma renin activity levels. On the other hand, during this treatment, urinary concentrations of the metabolites of cortisol, cortisone and corticosterone were only moderately affected.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1995

Mouse testis cell sorting according to DNA and mitochondrial changes during spermatogenesis.

Flow cytometry can measure variations in DNA content and chromatin structure as well as dramatic changes in the mitochondria of germ cells during maturation from spermatogonia to elongated spermatids. Using 10-N nonyl acridine orange (NAO), an inner mitochondrial membrane dye, it is easy to follow mitochondria rearrangements. Mouse testis cells stained with the DNA fluorescent probe propidium iodide (PI) and analyzed by flow cytometry can be discriminated on the basis of their ploidy levels into five main regions corresponding to elongated spermatids, round spermatids, diploid, S-phase, and tetraploid cells. The simultaneous use of PI and NAO demonstrated the presence of cells having low and high mitochondrial content in the haploid, diploid, and tetraploid compartments. Eleven sorting windows were selected from the bivariate analysis (PI/NAO) and the corresponding cells were identified by microscopic observation. Cells were also discriminated by two parameter analysis of DNA content vs. cell diameter. The definition of seven different regions allowed us to determine NAO or rhodamine 123 (Rh 123) uptakes in each compartment. We observed that the ratio (Rh 123/NAO) dramatically changed according to the progression of cell differentiation which occurs during spermatogenesis.

Acridine Orange↗
PubMed1995

Direct cardiolipin assay in yeast using the red fluorescence emission of 10-N-nonyl acridine orange.

The dye 10-N-nonyl-3,6-bis(dimethylamino)acridine (10-N-nonyl acridine orange) has been recently identified as a specific probe for cardiolipin (Ka = 2 x 10(6) M-1). It also interacts, at lower affinity (Ka = 7 x 10(4) M-1), with other acidic phospholipids [Petit, J. M., Maftah, A., Ratinaud, M. H. & Julien, R. (1992) Eur. J. Biochem. 209, 267-273]. In order to reduce the interference corresponding to monoacidic phospholipid binding, we have quantified cardiolipin by using a fluorimetric method based on the red fluorescence of the dye dimers formed at the diacidic phospholipid contact. Hence we have demonstrated that: (a) in yeast, the mitochondrion is the target of the dye whatever the cell metabolism; (b) membrane or protein organization and fatty acid unsaturation do not significantly modify the binding of 10-N-nonyl acridine orange. Using thin-walled vesicles, a linear relationship was established between the amount of cardiolipin and the red fluorescence emitted by the dye. Low red fluorescences were also observed with vesicles containing phosphatidylserine and phosphatidylinositol. However, at the same acidic phospholipid concentration, the fluorescence was much higher using cardiolipin-containing vesicles (fivefold that observed with phosphatidylserine-containing vesicles). Thus, 10-N-nonyl acridine orange was applied to cardiolipin quantification in yeast. This new method revealed that cells growing with a high glucose concentration contained 2.2 +/- 0.3 nmol cardiolipin/10(6) cells, whereas with lactate they contained about twice this amount (3.9 +/- 0.3 nmol cardiolipin).

Acridine Orange↗
PubMed1995

Endo-N-acetyl-beta-D-glucosaminidase and peptide-N4-(N-acetyl-glucosaminyl) asparagine amidase activities during germination of Raphanus sativus.

Endo-N-acetyl-beta-D-glucosaminidase (ENGase, EC 3.2.1.96) and peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase (PNGase, EC 3.5.1.52) activities were monitored during germination and postgerminative development in Raphanus sativus. The PNGase activity was found in dry seeds and its level was constant during germination and postgermination. The ENGase activity was first detected about 18 hr after the start of imbibition (HAI) and displayed a maximum level at 36 HAI. After 36 HAI the production of both enzymes was constant until days 4-5. Both enzymes displayed substrate specificities corresponding to the potential glycoprotein substrates found in plants. They are in agreement (i) with the hypothesis that ENGase and PNGase are at the origin of the production of 'unconjugated N-glycans' and (ii) with the possibility that protein activity could be regulated by the removal of N-glycans.

Amidohydrolases↗
PubMed1995

Do de-N-glycosylation enzymes have an important role in plant cells?

In this review de-N-glycosylation was defined as the removal of the glycan(s) from a N-glycosylprotein, by means of enzymes acting on the di-N-acetylchitobiosyl part of the invariant pentasaccharide inner-core of N-glycosylproteins. Peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidases (PNGase) and endo-N-acetyl-beta-D-glucosaminidases (ENGase) were both considered as de-N-glycosylation enzymes. A detailed description of the characterization and the function of plant PNGases and ENGases is presented, together with a brief presentation on the occurrence and the current knowledge on the function of microbial and animal enzymes. De-N-glycosylation of plant glycoproteins was proposed as a possible mechanism for the release of oligosaccharides displaying biological activities and the removal of N-glycans could also explain the regulation of protein activity. Each enzyme seems to have a specific function during germination and post-germinative development. All the arguments concur that de-N-glycosylation enzymes have an important role in plant cells and confirm that the N-glycosylation/de-N-glycosylation system should occur more commonly than presently recognized in living organisms.

Amidohydrolases↗
PubMed1995

Use of porcine fibrinogen as a model glycoprotein to study the binding specificity of the three variants of K88 lectin.

Known glycoproteins were used to determine the differences occurring in the binding specificities of the three variants of the K88 lectin in an approach essentially based on lectin blotting. During the screening, it was demonstrated that each variant of the K88 lectin biotinylated via its amino groups (NbioK88) exhibited a characteristic binding to the three chains of porcine fibrinogen. NbioK88ab weakly bound to A alpha chains, NbioK88ac bound to B beta and gamma chains, and NbioK88ad bound only to the gamma chain. To validate this model, the oligosaccharide moieties of porcine fibrinogen were analyzed with glycosidases and by lectin blotting and sugar composition. Both the B beta chain and gamma chain carry biantennary N-glycans of the N-acetyllactosamine type that are not recognized by K88 lectins. A alpha chains are substituted by sialylated T antigen. O-glycans were also detected on B beta and gamma chains of porcine fibrinogen and contribute to the recognition of these chains by K88ac and K88ad fimbriae.

Animals↗
PubMed1995

An endo-N-acetyl-beta-D-glucosaminidase, acting on the di-N-acetylchitobiosyl part of N-linked glycans, is secreted during sporulation of Myxococcus xanthus.

After the demonstration that Stigmatella aurantiaca DW4 secretes an endo-N-acetyl-beta-D-glucosaminidase (ENGase), acting on the di-N-acetylchitobiosyl part of N-linked glycans (S. Bourgerie, Y. Karamanos, T. Grard, and R. Julien, J. Bacteriol. 176:6170-6174, 1994), an ENGase activity having the same substrate specificity was also found to be secreted during vegetative growth of Myxococcus xanthus DK1622. The activity decreased in mutants known to secrete less protein than the wild type (Exc +/-). During submerged development, the activity was produced in two steps: the first increase occurred during the aggregation phase, and the second one occurred much later, during spore formation. This production was lower in developmental mutants impairing cell-cell signaling, the late mutants (csg and dsg) being the most deficient. Finally, when sporulation was obtained either by starvation in liquid shake flask culture or by glycerol induction, the activity was produced exclusively by the wild-type cells during the maturation of the coat.

Disaccharides↗
PubMed1995

Flow cytometric analysis of human epidermal cell ageing using two fluorescent mitochondrial probes.

Cardiolipin, mitochondrial transmembrane potential, cell refringence and cell diameter were examined in epidermal cells obtained from 42 women between 9- to 75-year-old. The study was carried out in situ by flow cytometry on cells having incorporated either Nonyl Acridine Orange or Rhodamine 123, 2 mitochondria-specific dyes. Cardiolipin levels, determined by the binding of the cardiolipin-specific probe Nonyl Acridine Orange, decreased significantly with age, especially in young individuals. This suggests 2 stages in the age-dependent transformation of mitochondria (organelle number and/or size): one during childhood development and to adulthood (9 to 27 years) in which cardiolipin levels decrease dramatically (slope: -3.742; p = 0.0243) and the other corresponding to senescence (35 to 75 years) in which this decrease is less pronounced (slope: -0.618; p = 0.0467). These changes have no effect on mitochondrial potential, measured by Rhodamine 123 incorporation, which remained constant with age. This function, controlling calcium partitioning within the cell, might allow keratinocytes to differentiate and maintain the skin barrier function of the epidermis. Like cardiolipin, intrinsic parameters such as cell size and refringence also significantly decreased in epidermal cells from elderly subjects. The methodology can be used to determine physiological ageing in various cell types and to analyse human ageing and related parameters.

Acridine Orange↗